Porcine circovirus (PCV) is a small, non-enveloped DNA virus which belongs to the family Circoviridae (Todd, D. et al., 2005, Circoviridae, p. 327-334. In C. M. Fauquet et al (ed.), Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, Elsevier Academic Press, San Diego). Type 1 PCV (PCV1) was discovered as a contaminant of the porcine kidney PK-15 cell line in the mid-seventies (Tischer, I. et al., 1974, Characterization of papovavirus- and picornavirus-like particles in permanent pig kidney cell lines, Zentralbl Bakteriol Orig A 226:153-67).
PCV1 was considered to be a non-pathogenic virus because inoculation of pigs with the PK-15 cell line-derived PCV1 virus did not cause any disease in pigs (Tischer, I. et al., 1986, Studies on epidemiology and pathogenicity of porcine circovirus, Arch Viroi 91:271-6). In 1997, a variant strain of PCV, designated PCV type-2 (PCV2), was discovered in piglets with wasting disease in Canada (Allan, G. M. et al., 1998, Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J Vet Diagn Invest 10:3-10; Clark, E. G., 1997, Presented at the 28th Annual Meeting of the American Association of Swine Practitioners; Ellis, J. et al., 1998, Isolation of circovirus from lesions of pigs with postweaning muitisystemic wasting syndrome, Can Vet J 39:44-51; Meehan, B. M. et al., 1998, Characterization of novel circovirus DNAs associated with wasting syndromes in pigs, J Gen Virol 79 (Pt 9):2171-9). Currently, PCV2 is the primary causative agent of porcine circovirus-associated disease (PCVAD), which includes wasting, mortality, respiratory signs, enteritis, reproductive failure, and porcine dermatitis and nephropathy syndrome (PDNS) (Opriessnig, T. et al., 2007, Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies, J Vet Diagn Invest 19:591-615). PCV2 is currently considered to be one of the most economically-important viral pathogens in global pig populations, and is found in every major swine producing country of the world (Gillespie, J. et al, 2009, Porcine Circovirus Type 2 and Porcine Circovirus-Associated Disease, J Vet Intern Med). Observation of severe clinical PCVAD in conventional pigs experimentally infected with PCV2 alone is uncommon, and coinfection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV) or porcine parvovirus (PPV) is usually required to induce the full-spectrum of clinical PCVAD (Allan, G. M. et al., 2000, Experimental infection of colostrum deprived piglets with porcine circovirus 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication, Arch Virol 145:2421-9; Opriessnig, T. et al. 2007. Supra; Roca, M. et al., 2004, In vitro and in vivo characterization of an infectious clone of a European strain of porcine circovirus type 2, J Gen Virol 85:1259-66; Rovira, A. et al., 2002, Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2, J Virol 76:3232-9; Tomas, A. et al., 2008, A meta-analysis on experimental infections with porcine circovirus type 2 (PCV2), Vet Microbiol 132:260-73). However, infection of caesarean-derived, colostrum-deprived (CD/CD) pigs with PCV2 alone has resulted in severe clinical PCVAD and mortality (Allan, G. et al. 2003, Reproduction of postweaning muitisystemic wasting syndrome in pigs experimentally inoculated with a Swedish porcine circovirus 2 isolate, J Vet Diagn Invest 15:553-60; Allan, G. M. et al., 2004, PMWS: experimental model and co-infections, Vet Microbiol 98:165-8; Bolin, S. R. et al., 2001, Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type 2 porcine circovirus, J Vet Diagn Invest 13:185-94; Harms, P. A. et al., 2001, Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus, Vet Pathol 38:528-39; Kennedy, S. et al., 2000, Reproduction of lesions of postweaning muitisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus, J Comp Pathol 122:9-24). Several comprehensive reviews of the pathogenesis, immunology, and molecular biology of PCV2 are available (Allan, G. M. and J. A. Ellis, 2000, Porcine circoviruses: a review. J Vet Diagn Invest 12:3-14; Ellis, J. et al., 2004, Porcine circovirus-2 and concurrent infections in the field, Vet Microbiol 98:159-63; Finsterbusch, T. and A. Mankertz, 2009, Porcine circoviruses-small but powerful, Virus Res 143:177-83; Gillespie, J. et al., 2009, supra; Mankertz, A. et al., 2004, Molecular biology of Porcine circovirus: analyses of gene expression and viral replication, Vet Microbiol 98:81-8; Opriessnig, T. et al. 2007, Supra; Ramamoorthy, S. and X. J. Meng, 2009, Porcine circoviruses: a minuscule yet mammoth paradox, Anim Health Res Rev 10:1-20; Segales, J. et al., 2005, Porcine circovirus diseases, Anim Health Res Rev 6:119-42).
Although the genomic organization of the pathogenic PCV2 and the non-pathogenic PCV1 is similar, the genomes of PCV1 and PCV2 share only approximately 68-76% nucleotide sequence identity (Fenaux, M. et al., 2004, Detection and in vitro and in vivo characterization of porcine circovirus DNA from a porcine-derived commercial pepsin product, J Gen Virol 85:3377-82; Hamel, A. L et al., 1998, Nucleotide sequence of porcine circovirus associated with postweaning muitisystemic wasting syndrome in pigs, J Virol 72:5262-7; Tischer, I. et al., 1982, A very small porcine virus with circular single-stranded DNA, Nature 295:64-6) and differences in transcriptional patterns and antigenic profile of the capsid protein have been reported (Cheung, A. K. 2003, Comparative analysis of the transcriptional patterns of pathogenic and nonpathogenic porcine circoviruses, Virology 310:41-9; Lekcharoensuk, P. et al., 2004, Epitope mapping of the major capsid protein of type 2 porcine circovirus (PCV2) by using chimeric PCV1 and PCV2, J Virol 78:8135-45; Shang, S. B. et al., 2009, Fine mapping of antigenic epitopes on capsid proteins of porcine circovirus, and antigenic phenotype of porcine circovirus type 2, Mol Immunol 46:327-34). The two major genes encoded by the viral genome include the 942 bp replicase (rep) gene (Mankertz, A. and B. Hillenbrand, 2001, Replication of porcine circovirus type 1 requires two proteins encoded by the viral rep gene, Virology 279:429-38) and the 702 bp capsid gene (cap) (Nawagitgul, P. et al., 2000, Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein, J Gen Virol 81:2281-7). The rep gene is highly conserved between PCV1 and PCV2 with about 83% nucleotide sequence identity while the cap gene shares only about 67-70% identity (Mankertz, A. et al., 2004, supra). Currently, at least three subtypes of PCV2 have been identified in swine herds worldwide: PCV2a, PCV2b, and PCV2c (Dupont, K. et al., 2008, Genomic analysis of PCV2 isolates from Danish archives and a current PMWS case-control study supports a shift in genotypes with time, Vet Microbiol 128:56-64; Segales, J. et al., 2008, PCV-2 genotype definition and nomenclature, Vet Rec 162:867-8). PCV2a and PCV2b have both been associated with clinical PCVAD of varying degrees of severity (An, D. J. et al., 2007, Phylogenetic characterization of porcine circovirus type 2 in PMWS and PDNS Korean pigs between 1999 and 2006, Virus Res 129:115-22; Ciacci-Zanella, J. R. et al., 2009, Detection of porcine Circovirus type 2 (PCV2) variants PCV2-1 and PCV2-2 in Brazilian pig population, Res Vet Sci 87:157-60; Lager, K. M et al., 2007, Mortality in pigs given porcine circovirus type 2 subgroup 1 and 2 viruses derived from DNA clones, Vet Rec 161:428-9; Madson, D. M. et al., 2008, Characterization of shedding patterns of Porcine circovirus types 2a and 2b in experimentally inoculated mature boars, J Vet Diagn invest 20:725-34; Opriessnig, T. et al., 2006, Genetic and experimental comparison of porcine circovirus type 2 (PCV2) isolates from cases with and without PCV2-associated lesions provides evidence for differences in virulence, J Gen Virol 87:2923-32; Opriessnig, T. et al., 2008, Differences in virulence among porcine circovirus type 2 isolates are unrelated to cluster type 2a or 2b and prior infection provides heterologous protection, J Gen Virol 89:2482-91). Prior to 2005, only PCV2a was found within pig populations in the United States and Canada, while both PCV2a and PCV2b were present in Europe and China (Chae, J. S. and K. S. Choi, 2009, Genetic diversity of porcine circovirus type 2 from pigs in Republic of Korea, Res Vet Sci; Dupont, K. et al., 2008, supra). Since 2005, novel PCV2b strains were recognized in the United States and there has been a global shift in a dominant prevalence of PCV2b in pig populations, concurrently with increased severity of clinical PCVAD (Carman, S. et al., 2008, The emergence of a new strain of porcine circovirus-2 in Ontario and Quebec swine and its association with severe porcine circovirus associated disease-2004-2006, Can J Vet Res 72:259-68; Chae, J. S. and K. S. Choi, 2009, supra; Cheung, A. K. et al., 2007, Detection of two porcine circovirus type 2 genotypic groups in United States swine herds, Arch Virol 152:1035-44; Ciacci-Zanella, J. R. et al., 2009, supra; Dupont, K. et al., 2008, supra; Gagnon, C. A. et al., 2007, The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada, Can Vet J 48:811-9; Lipej, Z. et al., 2005, Postweaning muitisystemic wasting syndrome (PMWS) in pigs in Croatia: detection and characterisation of porcine circovirus type 2 (PCV2), Acta Vet Hung 53:385-96; Wang, F. et al., 2009, Genetic variation analysis of Chinese strains of porcine circovirus type 2, Virus Res 145:151-6; Wiederkehr, D. D. et al., 2009, A new emerging genotype subgroup within PCV-2b dominates the PMWS epizooty in Switzerland, Vet Microbiol 136:27-35). The pathogenicity of PCV2c is unclear, as it has only been reported in non-diseased herds in Denmark in 1980, 1987, and 1990 (Dupont, K. et al., 2008, supra).
The current available commercial vaccines are all killed or recombinant vaccines based upon the PCV2a subtype (Opriessnig, T. et al. 2007, Supra; Ramamoorthy, S. and X. J. Meng, 2009, supra). The inventors have previously successfully developed an inactivated vaccine, Suvaxyn PCV2® One Dose™, based upon the PCV1-2a chimeric virus (with the capsid gene of PCV2a in the backbone of PCV1) (Fenaux, M. et al., 2004A, A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs, J Virol 78:6297-303; Fenaux, M. et al., 2003, Immunogenicity and pathogenicity of chimeric infectious DNA clones of pathogenic porcine circovirus type 2 (PCV2) and nonpathogenic PCV1 in weanling pigs, J Virol 77:11232-43; Gillespie, J. et al., 2008, A genetically engineered chimeric vaccine against porcine circovirus type 2 (PCV2) is genetically stable in vitro and in vivo, Vaccine 26:4231-6). However, since the PCV2b subtype has now become the globally dominant genotype associated with severe clinical PCVAD in commercial pigs, and since PCV2a and PCV2b differ by as much as 10% nucleotide sequence identity (Fenaux, M. et al., 2000, Genetic characterization of type 2 porcine circovirus (PCV-2) from pigs with postweaning muitisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2, J Clin Microbiol 38:2494-503; Olvera, A. et al., 2007, Molecular evolution of porcine circovirus type 2 genomes: phylogeny and clonality, Virology 357:175-85), it is unknown whether the current PCV2a subtype-based killed or recombinant vaccines provide complete protection against the newly-recognized PCV2b subtype. Several studies have demonstrated effectiveness of current commercial vaccines against PCV2b challenge (Fort, M. et al., 2008, Porcine circovirus type 2 (PCV2) vaccination of conventional pigs prevents viremia against PCV2 isolates of different genotypes and geographic origins, Vaccine 26:1063-71; Fort, M. et al., 2009, One dose of a porcine circovirus 2 (PCV2) sub-unit vaccine administered to 3-week-old conventional piglets elicits cell-mediated immunity and significantly reduces PCV2 viremia in an experimental model, Vaccine 27:4031-7; Opriessnig, T. et al., 2009, Comparison of efficacy of commercial one dose and two dose PCV2 vaccines using a mixed PRRSV-PCV2-SIV clinical infection model 2-3-months post vaccination, Vaccine 27:1002-7), however it is imperative to develop a PCV2b subtype-based vaccine, preferably a live-attenuated vaccine, against PCVAD. A live-attenuated vaccine based on the new PCV2b subtype would possibly provide much greater protection in the field than the current available killed and subunit vaccines based on the PCV2a subtype.